An important type of ionization for biomolecules is ionization by matrix assisted laser desorption (MALDI), which was developed about 20 years ago by M. Karas and K. Hillenkamp. MALDI ionizes the biomolecules, which are present at high dilution in a mixture with molecules of a matrix substance in samples on sample supports, by firing laser light pulses at them.
MALDI is in competition with electrospray ionization (ESI), which ionizes analyte molecules dissolved in a liquid, and which can therefore easily be coupled with separation procedures such as liquid chromatography or capillary electrophoresis. Although at present more mass spectrometers are equipped with electrospray ion sources than with MALDI ion sources, the development of modern laser and preparation techniques provides MALDI with a number of advantages over ESI. Hundreds of samples can be placed on one sample support. Pipetting robots are available for this purpose. The transport of a neighboring sample on the sample support into the focal point of a UV pulse laser takes a mere fraction of a second; as much time as necessary is then available for the various kinds of analytic methods that may be applied to this sample, limited only by complete consumption of the sample. This distinguishes MALDI from electrospray ionization, which offers slow sample changeover and which, when coupled with chromatography, limits the analysis time to the duration of the chromatographic peak. In addition, MALDI supplies only singly protonated molecule ions even from very heavy analyte molecules, a feature that makes the analysis of biomolecule mixtures easy compared with the wide variety of multiply protonated molecule ions delivered by ESI.
The use of MALDI to analyze peptides that have been separated by liquid chromatography and applied to MALDI sample supports is gaining ground (“HPLC-MALDI”). Also of interest is the use of MALDI in the imaging mass spectroscopy of thin histologic sections, a technique with which the spatial distribution of individual proteins or of specific pharmaceutical agents or their metabolites can be measured. Another application of MALDI is the identification of microbes on the basis of their protein profiles, and this is rapidly gaining popularity due to the high speed of the analysis and the outstanding accuracy of the identifications.
MALDI is particularly well suited to the identification of tryptically digested proteins that are first separated by 2D gel electrophoresis or other methods, and whose separated fractions are then processed to form separate MALDI samples. Suitable robots are available for the processing. The mass spectra of the digest mixtures show almost exclusively singly protonated digest molecules, whose masses can be determined precisely in appropriate mass spectrometers. From this, the original proteins can be determined by commercially available computer programs with the aid of protein databases.
For further characterizations of these digest peptides or other proteins, e.g., in respect of sequence errors or post-translational modifications (PTM), MALDI also offers two methods for generating and measuring the daughter ions of selected parent ions. One method is based on spontaneous fragmentation, for example in-source decay (ISD), which primarily delivers c and z fragment ions, while retaining all the bonds to PTM side-chains. The other method, post-source decay (PSD), in contrast, is based on “ergodic” (or “thermal”) fragmentation, which primarily yields b and y fragment ions of the amino acid backbone alone, with the loss of all the side-chains. For the purpose of structural analysis, the ability to acquire both kinds of daughter ion spectra from the same sample is extremely valuable, since a comparison of the two allows both the sequence of amino acids and the positions and masses of the side-chains (PTM) to be read. In addition, MALDI offers the option of further fragmenting ISD fragment ions, whereby the “granddaughter ion spectra” yield information about the structures of specific modification groups, for instance about the polysaccharides of the glycosylations.
In the past, inexpensive UV nitrogen lasers have been used for MALDI. These deliver a laser light pulse lasting a few nanoseconds, and their light beams can be focused by lenses onto a spot of between about 50 and 200 micrometers in diameter. Since, through deliberate adjustment, the “focal spot” on the sample does not correspond to the true focal diameter of the laser light beam, it is better to use the terms “spot” and “spot diameter” here. Nitrogen lasers, however, have a short service life of only a few million laser light pulses, which is a serious obstacle for high-throughput analysis. Solid-state lasers, with a service life that is more than a thousand times longer, are often used, although these require special beam-shaping.
The ions created by each individual laser light pulse are still primarily accelerated axially into a time-of-flight path in MALDI time-of-flight mass spectrometers (MALDI TOF MS) designed specially for this purpose. After transiting the flight path, the ions impinge on a detector that measures the mass-dependent arrival time of the ions and their quantity, and saves the digitized measurements as the time-of-flight spectrum. Repetition frequencies for the laser light pulses were between 20 and 200 hertz, but today MALDI TOF mass spectrometers are available with light pulse frequencies of up to 2 kilohertz. Nowadays, however, time-of-flight mass spectrometers with orthogonal ion injection (OTOF) are also increasingly being equipped with MALDI ion sources, and these record mass spectra at repetition rates of between 5 and 10 kilohertz. In both types of mass spectrometers, detectors for the ion beams are used that include a special secondary electron multiplier (SEM) followed by a transient recorder. The transient recorder contains a fast analog-to-digital converter (ADC), working at between 2 and 4 Gigahertz, usually with only 8-bit resolution. The mass spectra can be up to 100 or even 200 microseconds long, therefore comprising 200,000 to 800,000 measurements. The measurements from several hundreds or thousands of time-of-flight ion spectra measured in sequence in this way are added to form a sum spectrum. This is processed for peak detection, and the list of time-of-flight peaks is converted by a calibration function into a list of the masses m and their intensities i. This list, or its graphical representation i=f(m), is what is referred to as the “mass spectrum”. The mass spectra from both types of mass spectrometers can achieve mass resolutions of R=m/Δm=20,000 to 50,000, where Δm is the half-height width of the ion peaks.
Acquiring a mass spectrum typically refers to acquiring hundreds or thousands of individual spectra and combining them into a sum spectrum, as described above. This applies equally to mass spectra from molecule ions and to daughter ion spectra.
When the term “mass of the ions”, or simply “mass”, is used in connection with ions, it generally indicates the ratio m/z of the mass m to the number z of elementary charges, i.e., the physical mass m of the ions divided by the dimensionless, absolute number z of the positive or negative elementary charges carried by the ion. The rather unfortunate term “mass-to-charge ratio” is often used for m/z, even though it has the dimension of a mass.
Matrix assisted laser desorption uses (with a few exceptions) solid sample preparations on a sample support. The samples include small crystals of the matrix substance mixed with a small proportion (e.g., about a hundredth of a percent) of molecules of the analyte substances. The analyte molecules are individually incorporated in the crystal lattice of the matrix crystals, or are located at the crystal boundaries. The samples prepared in this way are exposed to short UV laser light pulses. The duration of the pulse is usually a few nanoseconds, and depends on the laser being used. This generates vaporization plasma containing neutral molecules and ions of the matrix substance plus a few analyte ions. It is reasonable to assume that, at least in normal protein analysis, the vast majority of analyte ions are formed reactively in the dense plasma by proton transfer from the mostly acid matrix molecules to the analyte molecules. Over a period of a few hundred nanoseconds, the plasma expands into the surrounding vacuum, loses density quickly, and cools adiabatically, as a result of which all the plasma particles are inhibited from further reactions.
The ratio of analyte molecules to matrix molecules is usually one in 10,000 at most, which keeps the analyte molecules apart from each other, and thus dimer ions are not formed. However, the analyte substances can form a mixture in which concentration ratios covering several orders of magnitude may be found between the various analyte substances to be measured. Measuring the analyte molecules then requires the mass spectrometer to have a high dynamic measuring range. Because the dynamic measuring range of each individual mass spectrum recorded by the transient recorder is normally limited to 8 bits, i.e., to measurements extending from 1 to 256, the high dynamic measuring range can only be achieved by recording hundreds or thousands of single mass spectra.
In MALDI mass spectrometry, considerable skill is required to set the detector amplification and the MALDI conditions to optimally exploit the 8-bit dynamic range of the transient recorder without either exceeding this measurement range through oversaturation, or failing to discover a part of the ions as a result of a signal that is too weak. Since the distribution of secondary electrons from single impacts of ions on the secondary electron amplifier forms a Poisson distribution with a mean value of about 2 or 3 electrons, the amplification in the secondary electron amplifier is optimally adjusted if a single ion generates, on average, a signal of about 2.5 counts of the ADC in the transient recorder. The measurement range for ions that reach the detector within the measurement period of the ADC of 0.5 or 0.25 nanoseconds is then 1:100 (2.5 counts:256 counts). Since an ion signal for ions of the same mass extends over several measuring periods, there must not be more than a few hundred ions in an ion signal containing ions of the same mass, and this must be achieved by adjusting the MALDI conditions. Optimal adjustment of the MALDI conditions calls for a great deal of knowledge about the effect of the laser light parameters on the MALDI processes.
The matrix substances employed, including mostly aromatic acids, mean that one of the parameters for the laser light is already largely determined, i.e., the wavelength of UV light. Wavelengths of between 330 and 360 nm, which are well absorbed by the aromatic groups of the best known matrix substances, have proved to be successful. Nitrogen lasers deliver light with a wavelength λ of 337 nm, while the most widely used neodymium-YAG lasers have, with frequency tripling, a wavelength λ of 355 nm. Pulses of light of both these wavelengths appear to have very much the same effect on the MALDI process. The wavelength of the light and the absorption coefficient of the matrix substance determine the penetration depth of the laser radiation into the solid material of the matrix crystals. The intensity of the radiation as it penetrates the material falls off with a half-value depth of between a few tens and a few hundreds of a nanometer.
In addition to the UV wavelength and the penetration depth, there are three other important parameters that characterize the laser light pulse on the sample:
(1) the total energy of the laser light pulse, normally measured in microjoules (μJ);
(2) the energy density (fluence), which is the energy per unit area in the laser spot (or in multiple synchronously generated laser spots), measured, for instance, in nanojoules per square micrometer (nJ/μm2); and
(3) the power density on the surface of the sample, i.e., the energy density per unit of time, which is determined by the length of the laser light pulse. This can, for instance, be measured in nanojoules per square micrometer and nanosecond (nJ/(μm2×ns)). Our investigations have found the last two of these parameters to be particularly important: laser light pulses containing the same energy but with different durations do not have the same effect at all.
The detailed review article entitled “The Desorption Process in MALDI” by Klaus Dreisewerd (Chem. Rev. 2003, 103, 395-425) refers to papers reporting the effects of many parameters such as spot diameter, laser light pulse duration, and energy density on the desorption and the creation of matrix ions and analyte ions. Although the effects of many of these parameters are not independent from one another, hardly ever have all the parameters been carefully varied in relation to one another. It has been reported, for instance, that varying the laser pulse duration between 0.55 and 3.0 nanoseconds does not have any influence on the formation of the ions. The diameter of the spot, however, was not varied or even stated. For varying spot diameters, on the other hand, the threshold of the energy density for the first occurrence of ions has been investigated, yet without examining the profile of the energy density in the laser spot, which, according to our investigations, is of high significance. Moreover, according to this literature source, this threshold rises sharply as the spot diameter becomes smaller For example, a spot diameter of about 10 micrometers, something like 10 times the energy density (fluence) is required as for a spot diameter of 200 micrometers. We cannot confirm this for these spot diameters, even though a rise in the threshold energy density is to be expected for significantly smaller spot diameters, because for tiny spots too much energy can quickly flow away laterally to the surroundings. It appears that little is reported in the literature about how spot diameter and duration of laser pulse affect the kind of ionization, and particularly the fragmentation of the analyte molecules.
Previous investigations of the MALDI process were, however, impaired because the techniques used for preparing the samples were not reproducible. Generally speaking, droplets with dissolved matrix and analyte molecules were simply applied to the sample support plate and dried. These samples were highly inhomogeneous, and it was regularly necessary to search for “hot spots” containing analyte molecules on the sample to analyze these substances. A quantitative approach was out of the question. The majority of investigations of the MALDI process have been made with these samples, and this may explain many of the inconsistencies between these investigations.
In the meantime, it is possible to manufacture highly reproducible thin layers for a number of non-water-soluble matrix substances, such as α-cyano-4-hydroxycinnamic acid (CHCA), including just a single layer of closely packed crystals having a diameter of only about 1 micrometer. A predominantly aqueous solution of analyte molecules is then applied to this dry, thin layer of matrix crystals; the matrix crystals bind the analyte molecules superficially, without themselves dissolving. After about half a minute or one minute, the excess solvent can then be sucked off, which removes many contaminants such as salts. However, a proportion of excess analyte molecules may be removed at the same time, and this must be borne in mind for quantitative investigations. The superficially adsorbed analyte molecules can subsequently be embedded into the matrix crystals, after drying, by applying an organic solvent that begins to dissolve the matrix crystals. Once this solvent has evaporated, an extremely homogeneous sample is obtained. That is, at every location, with small statistical variations, it delivers the same ion currents with the same analytic results. Today, sample carrier plates to which thin layers of CHCA have already been applied are manufactured commercially. Adequate investigations have yet to be published regarding the MALDI processes that take place on these thin layer samples.
The article by Dreisewerd cited above, presents a number of interesting measurement curves. From the first appearance of analyte ions, the yield of analyte ions rises non-linearly over several orders of magnitude with about sixth or seventh power of the energy density of the laser radiation. These measurements, which have been confirmed a number of times, are very interesting. If we assume that the ablation of substance is proportional to the energy density, then the degree of ionization of analyte molecules, and therefore the utilization of the sample, should rise in proportion to this higher power of the energy density. It follows from this that by shrinking the laser spot whilst keeping the total energy of the laser light pulse constant, it should to be possible to increase the yield of analyte ions. Interestingly, this cannot be confirmed for nitrogen lasers, with which the majority of investigations are made. As our own investigations show, the reason for this is that the nitrogen laser does not have a homogeneous energy density profile; rather, in each laser pulse, there is just one, or a few, micro-spots of high energy density, whose position varies from one pulse to the next. When the spot diameter is reduced in size by focusing the laser light beam, the diameter of the micro-spots does not change, as these are at the limit of the focusing capacity of the lens system. The energy density in these micro-spots therefore does not change either. But the micro-spots have diameters that are below the requirement for optimal ion yield.
The situation is different with solid-state lasers. They deliver a smooth energy density profile across the laser spot provided by the lens system. The energy density profile has an approximately Gaussian distribution. The introduction of solid-state lasers into MALDI technology in place of the nitrogen lasers previously used led to the surprising discovery that the smooth beam profile from these solid-state lasers actually reduced the yield of ions from thin-layer preparations. According to our own investigations, the reason for this is that when the energy density is adjusted for optimal utilization of the dynamic measuring range of the ion detector, the ion yield is only a little above the threshold. For this reason, a technique for inhomogeneous beam profiling was developed, which increases the ion yield even beyond the ion yield obtained from nitrogen lasers. See for example, U.S. Pat. No. 7,235,781. It is thus possible to increase the ion yield by optimizing the number and diameter of the laser spots. By profiling the laser beam, a high yield of analyte ions, relative to the original number of analyte molecules on the sample, is achieved at the same time as optimal adaptation to the measuring range of the transient recorder.